CN103409395B - Method for fermenting microbe to prepare endo-chitosanase - Google Patents
Method for fermenting microbe to prepare endo-chitosanase Download PDFInfo
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Abstract
The invention discloses a method for fermenting a microbe to prepare endo-chitosanase, wherein the microbe is Streptomyces roseolus DH, and the fermentation method comprises the steps of strain activation, seed solution culture, enzyme production by fermentation of a fermentation tank and collection of crude enzyme. The fermentation method provided by the invention has the advantages that high-activity endo-chitosanase can be produced, the activity of the endo-chitosanase in fermentation liquor is 35 U/ml, the fermentation period is 50-60 hours, the crude enzyme can be directly applied to production of chitosan oligosaccharide, the fermentation process is simple, the condition is easy to control, the yield of enzyme activity is high, the fermentation period is short, and the fermentation liquor is simple to process; the method is applicable to industrial production of endo-chitosanase.
Description
Technical field
The present invention relates to fermentable and technical field of enzyme engineering, specifically, is a kind of method that fermentable prepares endo-type chitoanase.
Background technology
Oligochitosan refers to that the polymerization degree is the poly-glucosamine of 2 ~ 10, compare its precursor substance chitin and chitosan, except there is common good biocompatibility, also possesses following advantageous characteristic: good water solubility, easily absorbed by body and there is antibacterial, anti-tumour phological activity etc.Therefore, oligochitosan has broad application prospects in each fields such as medicine, food, agricultural, environment and water treatments.At present, application chitosan is prepared oligochitosan and is mainly contained chemical degradation method and enzyme liberating method, wherein, biological enzyme, because possessing that specificity is strong, reaction conditions is gentle, process is easy to control and the feature such as low in the pollution of the environment, is thought by those skilled in the art and is had better potentiality to be exploited.
Chitoanase (Chitosanase, EC3.2.1.132) can the hydrolysis of β-Isosorbide-5-Nitrae-glycosidic link in catalysis chitosan molecule in a mild condition, for enzyme process prepares the topmost limiting factor of oligochitosan.Chitoanase is mainly derived from the microorganism such as fungi, bacterium, and the chitoanase character of gained is not quite similar because of its source.Produce bacterial strain to acquired chitoanase and carry out deep enzymology, result shows, these microorganisms, when being subject to inducing, express two or more endo-type and/or circumscribed-type chitoanase simultaneously.Product after endo-type chitoanase thoroughly decomposes chitosan is mainly the chitooligosaccharide-that the polymerization degree is 2 ~ 4, and the enzymolysis product of circumscribed-type chitoanase is then shell monose, and namely the polymerization degree is 1.In current this area, the oligochitosan of indication mainly refers to that the polymerization degree is the chitooligosaccharide-of 2 ~ 10, due to circumscribed-type and endo-type chitoanase with existing, make the production polymerization degree be 2 ~ 10 oligochitosan become an insoluble problem.
Wei Fuwei etc. (referring to: the screening of chitoanase Producing Strain, qualification and enzymatic production pre-test, Shanghai Ocean University's journal, in March, 2011) sift out a strain Chitosan-Hydrolytic Bacterium, called after misty rose streptomycete
streptomyces roseolusdH, basic fermentation condition is after optimizing, and in fermented liquid, chitoanase vigor reaches 6.1U/mL.Basic fermentation condition is: by 2%(V/V) inoculum size access fermention medium carry out shake flask fermentation, leavening temperature 30 DEG C, rotating speed 150r/min, liquid amount 100mL/250mL, shaking table concussion cultivate 60h.But about fermentation process of the present invention, yet there are no report.
Summary of the invention
The object of the invention is for deficiency of the prior art, provide a kind of fermentable to prepare the method for endo-type chitoanase, in gained fermented liquid, the enzyme activity of chitoanase reaches 35U/mL.
For achieving the above object, the technical scheme that the present invention takes is:
Fermentable prepares a method for endo-type chitoanase, and described microorganism is misty rose streptomycete
streptomyces roseolusdH.Bacterial strain uses therefor of the present invention is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, and preserving number is CGMCC No.7742, preservation day is on June 18th, 2013, and Classification And Nomenclature is
streptomyces roseolus.Described fermentation process comprises the following steps:
(1) actication of culture: by the misty rose streptomycete preserved
streptomyces roseolusthe plate culture medium that it is sole carbon source that DH is inoculated in colloid chitosan, is inverted cultivation 2 ~ 4 days with activated strains in 30 DEG C of incubators; Described slat chain conveyor based formulas is: (NH
4)
2sO
40.51 ~ 1.0g, K
2hPO
40.21 ~ 0.5g, sodium-chlor 0.51 ~ 1.0g, magnesium sulfate 0.01 ~ 0.2g, colloid chitosan 1.01 ~ 1.2g, agar 2.0g, adds water to 100mL, pH 7.0 ~ 7.2;
(2) seed liquor is cultivated: the single colony inoculation after picking activation, in liquid seed culture medium, carries out shaking table and to spread cultivation bacterial strain, be cultured to the OD of seed liquor under 150r/min, 30 DEG C of conditions
600reach 0.8 ~ 1.0, stand-by; Described seed culture based formulas is: peptone 0.51 ~ 1.0g, glucose 0.21 ~ 0.5g, sodium-chlor 0.8 ~ 1.0g, K
2hPO
40.02 ~ 1.0g, KH
2pO
40.01 ~ 0.05g, yeast powder 0.2 ~ 1.0g, magnesium sulfate 0.02 ~ 0.08g, adds water to 100mL, natural pH;
(3) ferment tank produces enzyme: the liquid amount of fermention medium by volume per-cent 40% ~ 70% loads in fermentor tank, by the seed liquor in step (2) by volume per-cent 2.1% ~ 3% inoculum size access fermention medium in, described fermentor tank is flat-blade turbine stirring-type fermentor tank, tank pressure 0.08 ~ 0.11MPa is kept in fermenting process, stir speed (S.S.) is 180 ~ 250r/min, controls air velocity 1.2 ~ 2.0m
3/ h, leavening temperature is 30 ~ 31 DEG C, and fermentation time is 50 ~ 60h; Described fermentative medium formula is: colloid chitosan 0.9 ~ 1.2g, sodium-chlor 0.8 ~ 1.0g, K
2hPO
40.05 ~ 0.15g, KH
2pO
40.04 ~ 0.2g, magnesium sulfate 0.03 ~ 0.1g, yeast extract 0.2 ~ 2.0g, peptone 0.2 ~ 2.0g, adds water to 100mL, adjusts pH to 7.0 ~ 7.2 with 2mol/L hydrochloric acid;
(4) collection of crude enzyme liquid: closed cans discharging after fermentation, merges supernatant liquor after centrifugal fermented liquid, namely obtains the crude enzyme liquid of endo-type chitoanase.
The product of described endo-type chitoanase hydrolyzing chitosan to be the polymerization degree be 2 ~ 10 oligochitosan.
The preparation method of described colloid chitosan is: chitosan is dissolved in the HC1 solution of 0.1 ~ 2.0mol/L with the concentration of 1g/L, room temperature for overnight, the NaOH solution of volumetric molar concentration such as to add, be neutralized to pH 9.0 ~ 10.0, filter to obtain flocks with 300 ~ 400 order yarn thin,tough silk, and precipitate 1 ~ 3 time with water repeated washing, until pH is neutral, finally with the speed homogenized precipitation 8 ~ 15min of 10000r/mim, after beating, namely obtain colloid chitosan.
Slat chain conveyor based formulas in described step (1) is: (NH
4)
2sO
40.6g, K
2hPO
40.3g, sodium-chlor 0.52g, magnesium sulfate 0.1g, colloid chitosan 1.1g, agar 2.0g, adds water to 100mL, pH 7.0.
The OD of seed liquor in described step (2)
600be 0.9 ~ 1.0.
Seed culture based formulas in described step (2) is: peptone 0.6g, glucose 0.3g, sodium-chlor 0.8g, K
2hPO
40.07g, KH
2pO
40.03g, yeast powder 0.5g, magnesium sulfate 0.05g, adds water to 100mL, natural pH.
The volume of fermentor tank is 10 liters in described step (3), and the liquid amount of fermention medium by volume per-cent 60% loads fermentor tank.
The inoculum size access fermention medium of seed liquor by volume per-cent 3% in described step (3), described stir speed (S.S.) is 200r/min, and air velocity is 1.4m
3/ h, leavening temperature is 30.5 DEG C, and tank pressure is 0.1MPa.
Fermentative medium formula in described step (3) is: colloid chitosan 1.0g, sodium-chlor 0.8g, K
2hPO
40.07g, KH
2pO
40.04g, magnesium sulfate 0.05g, yeast extract 0.5g, peptone 0.5g, adds water to 100mL, adjusts pH to 7.2 with 2mol/L hydrochloric acid.
Centrifugal condition in described step (4) is centrifugal 10min under 3000r/min rotating speed.
The enzyme activity determination method of the endo-type chitoanase that the inventive method obtains is DNS method, it is an enzyme activity unit (U) that enzyme activity unit is defined as the per minute enzyme amount produced needed for 1 μm of ol glucosamine, concrete operations reference literature (refers to: the screening of chitoanase Producing Strain, qualification and enzymatic production pre-test, Shanghai Ocean University's journal, Wei Fuwei, Jiang Xiayun, Chen Shunsheng, Chen Daochun, party cultivates).
The inventive method advantage is:
1, fermentation process provided by the invention, can produce the endo-type chitoanase of high vigor, and in fermented liquid, endo-type chitoanase vigor reaches 35U/mL, and this enzyme liquid directly can apply to the production of oligochitosan, enormously simplify technique;
2, this zymotechnique is simple, and condition is easy to control, and enzyme yield alive is high, fermentation period 50 ~ 60 hours, and fermentation period is short, and fermentation liquor treatment is simple, is suitable for the suitability for industrialized production of endo-type chitoanase.
Embodiment
Below embodiment provided by the invention is elaborated.
In embodiments of the invention, agents useful for same is commercial goods.Bacterial strain is misty rose streptomycete
streptomyces roseolusdH.Fermentor tank model is SFY-10, volume 10 liters, and be flat-blade turbine stirring-type fermentor tank, production company is Zhenjiang Jiang great Science and Technology Ltd..In enzyme activity determination, instrument spectrophotometer model is the T6 new millennium, and production company is Beijing Pu Xi general finite company.
embodiment 1
The preparation of colloid chitosan: chitosan is dissolved in the HC1 solution of 0.2mol/L with the concentration of 1g/L, room temperature for overnight, the NaOH solution of volumetric molar concentration such as to add, be neutralized to pH 9.5, flocks is filtered to obtain with 350 order yarn thin,tough silk, and by water repeated washing precipitation until pH is neutral, finally with the speed homogenized precipitation 13min of 10000r/mim, after beating, namely obtain colloid chitosan.
In actication of culture, slat chain conveyor based formulas used is: (NH
4)
2sO
40.6g, K
2hPO
40.3g, sodium-chlor 0.52g, magnesium sulfate 0.1g, colloid chitosan 1.1g, agar 2.0g, adds water to 100mL, pH 7.0.121 DEG C, sterilizing 20 minutes, stand-by.
Liquid seed culture medium formula is: peptone 0.6g, glucose 0.3g, sodium-chlor 0.8g, K
2hPO
40.07g, KH
2pO
40.03g, yeast powder 0.5g, magnesium sulfate 0.05g, adds water to 100mL, natural pH.200mL substratum loads 500mL triangular flask, seals with 8 layers of gauze, tin mulberry paper, puts into the Autoclave sterilizing 20 minutes of 121 DEG C, stand-by.
Fermentative medium formula is: colloid chitosan 1.0g, sodium-chlor 0.8g, K
2hPO
40.07g, KH
2pO
40.04g, magnesium sulfate 0.05g, yeast extract 0.5g, peptone 0.5g, adds water to 100mL, adjusts pH to 7.2 with 2mol/L hydrochloric acid.
Fermenting process comprises the following steps:
(1) actication of culture: adopt the method that line is separated, by the misty rose streptomycete preserved
streptomyces roseolusthe plate culture medium that it is sole carbon source that DH is inoculated in colloid chitosan, is inverted cultivation 3 days with activated strains in 30 DEG C of incubators;
(2) seed liquor is cultivated: the single colony inoculation after picking activation, in liquid seed culture medium, carries out shaking table and to spread cultivation bacterial strain, return to zero, be cultured to the OD of seed liquor with nonvaccinated substratum under 150r/min, 30 DEG C of conditions
600reach 0.9 ~ 1.0, stand-by;
(3) ferment tank produces enzyme: the liquid amount of fermention medium by volume per-cent 60% loads in 10L fermentor tank, then the edible vegetable oil adding 10mL is as defoamer, is taken up in order of priority carries out that sky slake substratum is real to disappear by the using method of fermentor tank; By in the inoculum size access fermention medium of the by volume per-cent 3% of the seed liquor in step (2), keep tank pressure 0.1MPa in fermenting process, stir speed (S.S.) is 200r/min, controls air velocity 1.4m
3/ h, leavening temperature is 30.5 DEG C, and fermentation time is 53h;
(4) collection of crude enzyme liquid: closed cans discharging after fermentation, fermented liquid is centrifugal 10min under 3000r/min rotating speed, finally merges supernatant liquor, namely obtains the crude enzyme liquid containing endo-type chitoanase.
Carry out enzyme activity determination to the crude enzyme liquid of step (4) gained, result shows, in fermented liquid, the enzyme activity of endo-type chitoanase is 35.2U/mL.
embodiment 2
The preparation of colloid chitosan: chitosan is dissolved in the HC1 solution of 1.0mol/L with the concentration of 1g/L, room temperature for overnight, the NaOH solution of volumetric molar concentration such as to add, be neutralized to pH 9.0, flocks is filtered to obtain with 300 order yarn thin,tough silk, and by water repeated washing precipitation until pH is neutral, finally with the speed homogenized precipitation 15min of 10000r/mim, after beating, namely obtain colloid chitosan.
In actication of culture, slat chain conveyor based formulas used is: (NH
4)
2sO
40.51g, K
2hPO
40.21g, sodium-chlor 0.7g, magnesium sulfate 0.01g, colloid chitosan 1.01g, agar 2.0g, adds water to 100mL, pH 7.1.121 DEG C, sterilizing 20 minutes, stand-by.
Liquid seed culture medium formula is: peptone 0.51g, glucose 0.21g, sodium-chlor 0.9g, K
2hPO
40.02g, KH
2pO
40.01g, yeast powder 0.2g, magnesium sulfate 0.02g, adds water to 100mL, natural pH.200mL substratum loads 500mL triangular flask, seals with 8 layers of gauze, tin mulberry paper, puts into the Autoclave sterilizing 20 minutes of 121 DEG C, stand-by.
Fermentative medium formula is: colloid chitosan 0.9g, sodium-chlor 0.9g, K
2hPO
40.05g, KH
2pO
40.1g, magnesium sulfate 0.03g, yeast extract 0.2g, peptone 0.2g, adds water to 100mL, adjusts pH to 7.0 with 2mol/L hydrochloric acid.
Fermenting process comprises the following steps:
(1) actication of culture: adopt the method that line is separated, by the misty rose streptomycete preserved
streptomyces roseolusthe plate culture medium that it is sole carbon source that DH is inoculated in colloid chitosan, is inverted cultivation 2.5 days with activated strains in 30 DEG C of incubators;
(2) seed liquor is cultivated: the single colony inoculation after picking activation, in liquid seed culture medium, carries out shaking table and to spread cultivation bacterial strain, return to zero, be cultured to the OD of seed liquor with nonvaccinated substratum under 150r/min, 30 DEG C of conditions
600reach 0.8 ~ 0.9, stand-by;
(3) ferment tank produces enzyme: the liquid amount of fermention medium by volume per-cent 40% loads in 10L fermentor tank, then the edible vegetable oil adding 8mL is as defoamer, is taken up in order of priority carries out that sky slake substratum is real to disappear by the using method of fermentor tank; By in the inoculum size access fermention medium of the by volume per-cent 2.1% of the seed liquor in step (2), keep tank pressure 0.08 MPa in fermenting process, stir speed (S.S.) is 180r/min, controls air velocity 1.2m
3/ h, leavening temperature is 30 DEG C, and fermentation time is 50h;
(4) collection of crude enzyme liquid: closed cans discharging after fermentation, fermented liquid is centrifugal 12min under 3000r/min rotating speed, finally merges supernatant liquor, namely obtains the crude enzyme liquid containing endo-type chitoanase.
Carry out enzyme activity determination to the crude enzyme liquid of step (4) gained, result shows, in fermented liquid, the enzyme activity of endo-type chitoanase is 22.1U/mL.
embodiment 3
The preparation of colloid chitosan: chitosan is dissolved in the HC1 solution of 0.1mol/L with the concentration of 1g/L, room temperature for overnight, the NaOH solution of volumetric molar concentration such as to add, be neutralized to pH 10.0, flocks is filtered to obtain with 400 order yarn thin,tough silk, and by water repeated washing precipitation until pH is neutral, finally with the speed homogenized precipitation 8min of 10000r/mim, after beating, namely obtain colloid chitosan.
In actication of culture, slat chain conveyor based formulas used is: (NH
4)
2sO
40.8g, K
2hPO
40.25g, sodium-chlor 0.8g, magnesium sulfate 0.05g, colloid chitosan 1.05g, agar 2.0g, adds water to 100mL, pH 7.0.121 DEG C, sterilizing 20 minutes, stand-by.
Liquid seed culture medium formula is: peptone 0.65g, glucose 0.25g, sodium-chlor 0.85g, K
2hPO
40.05g, KH
2pO
40.02g, yeast powder 0.6g, magnesium sulfate 0.07g, adds water to 100mL, natural pH.200mL substratum loads 500mL triangular flask, seals with 8 layers of gauze, tin mulberry paper, puts into the Autoclave sterilizing 20 minutes of 121 DEG C, stand-by.
Fermentative medium formula is: colloid chitosan 1.1g, sodium-chlor 0.85g, K
2hPO
40.1g, KH
2pO
40.08g, magnesium sulfate 0.06g, yeast extract 0.8g, peptone 0.8g, adds water to 100mL, adjusts pH to 7.1 with 2mol/L hydrochloric acid.
Fermenting process comprises the following steps:
(1) actication of culture: adopt the method that line is separated, by the misty rose streptomycete preserved
streptomyces roseolusthe plate culture medium that it is sole carbon source that DH is inoculated in colloid chitosan, is inverted cultivation 4 days with activated strains in 30 DEG C of incubators;
(2) seed liquor is cultivated: the single colony inoculation after picking activation, in liquid seed culture medium, carries out shaking table and to spread cultivation bacterial strain, return to zero, be cultured to the OD of seed liquor with nonvaccinated substratum under 150r/min, 30 DEG C of conditions
600reach 0.9 ~ 1.0, stand-by;
(3) ferment tank produces enzyme: the liquid amount of fermention medium by volume per-cent 50% loads in 10L fermentor tank, then the edible vegetable oil adding 9mL is as defoamer, is taken up in order of priority carries out that sky slake substratum is real to disappear by the using method of fermentor tank; By in the inoculum size access fermention medium of the by volume per-cent 2.5% of the seed liquor in step (2), keep tank pressure 0.11MPa in fermenting process, stir speed (S.S.) is 220r/min, controls air velocity 1.6m
3/ h, leavening temperature is 30.4 DEG C, and fermentation time is 55h;
(4) collection of crude enzyme liquid: closed cans discharging after fermentation, fermented liquid is centrifugal 10min under 4000r/min rotating speed, finally merges supernatant liquor, namely obtains the crude enzyme liquid containing endo-type chitoanase.
Carry out enzyme activity determination to the crude enzyme liquid of step (4) gained, result shows, in fermented liquid, the enzyme activity of endo-type chitoanase is 31.7U/mL.
embodiment 4
The preparation of colloid chitosan: chitosan is dissolved in the HC1 solution of 1.5mol/L with the concentration of 1g/L, room temperature for overnight, the NaOH solution of volumetric molar concentration such as to add, be neutralized to pH 9.5, flocks is filtered to obtain with 300 order yarn thin,tough silk, and by water repeated washing precipitation until pH is neutral, finally with the speed homogenized precipitation 14min of 10000r/mim, after beating, namely obtain colloid chitosan.
In actication of culture, slat chain conveyor based formulas used is: (NH
4)
2sO
41.0g, K
2hPO
40.4g, sodium-chlor 0.6g, magnesium sulfate 0.15g, colloid chitosan 1.15g, agar 2.0g, adds water to 100mL, pH 7.2.121 DEG C, sterilizing 20 minutes, stand-by.
Liquid seed culture medium formula is: peptone 0.55g, glucose 0.4g, sodium-chlor 1.0g, K
2hPO
40.1g, KH
2pO
40.04g, yeast powder 0.8g, magnesium sulfate 0.08g, adds water to 100mL, natural pH.200mL substratum loads 500mL triangular flask, seals with 8 layers of gauze, tin mulberry paper, puts into the Autoclave sterilizing 20 minutes of 121 DEG C, stand-by.
Fermentative medium formula is: colloid chitosan 1.2g, sodium-chlor 1.0g, K
2hPO
40.08g, KH
2pO
40.2g, magnesium sulfate 0.08g, yeast extract 1.0g, peptone 1.0g, adds water to 100mL, adjusts pH to 7.0 with 2mol/L hydrochloric acid.
Fermenting process comprises the following steps:
(1) actication of culture: adopt the method that line is separated, by the misty rose streptomycete preserved
streptomyces roseolusthe plate culture medium that it is sole carbon source that DH is inoculated in colloid chitosan, is inverted cultivation 3 days with activated strains in 30 DEG C of incubators;
(2) seed liquor is cultivated: the single colony inoculation after picking activation, in liquid seed culture medium, carries out shaking table and to spread cultivation bacterial strain, return to zero, be cultured to the OD of seed liquor with nonvaccinated substratum under 150r/min, 30 DEG C of conditions
600reach 0.9 ~ 1.0, stand-by;
(3) ferment tank produces enzyme: the liquid amount of fermention medium by volume per-cent 70% loads in 10L fermentor tank, then the edible vegetable oil adding 10mL is as defoamer, is taken up in order of priority carries out that sky slake substratum is real to disappear by the using method of fermentor tank; By in the inoculum size access fermention medium of the by volume per-cent 3% of the seed liquor in step (2), keep tank pressure 0.1MPa in fermenting process, stir speed (S.S.) is 240r/min, controls air velocity 1.8m
3/ h, leavening temperature is 30.6 DEG C, and fermentation time is 54h;
(4) collection of crude enzyme liquid: closed cans discharging after fermentation, fermented liquid is centrifugal 10min under 4000r/min rotating speed, finally merges supernatant liquor, namely obtains the crude enzyme liquid containing endo-type chitoanase.
Carry out enzyme activity determination to the crude enzyme liquid of step (4) gained, result shows, in fermented liquid, the enzyme activity of endo-type chitoanase is 28.5U/mL.
embodiment 5
The preparation of colloid chitosan: chitosan is dissolved in the HC1 solution of 2.0mol/L with the concentration of 1g/L, room temperature for overnight, the NaOH solution of volumetric molar concentration such as to add, be neutralized to pH 9.0, flocks is filtered to obtain with 350 order yarn thin,tough silk, and by water repeated washing precipitation until pH is neutral, finally with the speed homogenized precipitation 10min of 10000r/mim, after beating, namely obtain colloid chitosan.
In actication of culture, slat chain conveyor based formulas used is: (NH
4)
2sO
40.7g, K
2hPO
40.5g, sodium-chlor 1.0g, magnesium sulfate 0.2g, colloid chitosan 1.2g, agar 2.0g, adds water to 100mL, pH 7.1.121 DEG C, sterilizing 20 minutes, stand-by.
Liquid seed culture medium formula is: peptone 0.8g, glucose 0.5g, sodium-chlor 0.9g, K
2hPO
40.5g, KH
2pO
40.03g, yeast powder 1.0g, magnesium sulfate 0.04g, adds water to 100mL, natural pH.200mL substratum loads 500mL triangular flask, seals with 8 layers of gauze, tin mulberry paper, puts into the Autoclave sterilizing 20 minutes of 121 DEG C, stand-by.
Fermentative medium formula is: colloid chitosan 1.05g, sodium-chlor 0.95g, K
2hPO
40.15g, KH
2pO
40.15g, magnesium sulfate 0.1g, yeast extract 2.0g, peptone 2.0g, adds water to 100mL, adjusts pH to 7.1 with 2mol/L hydrochloric acid.
Fermenting process comprises the following steps:
(1) actication of culture: adopt the method that line is separated, by the misty rose streptomycete preserved
streptomyces roseolusthe plate culture medium that it is sole carbon source that DH is inoculated in colloid chitosan, is inverted cultivation 2 days with activated strains in 30 DEG C of incubators;
(2) seed liquor is cultivated: the single colony inoculation after picking activation, in liquid seed culture medium, carries out shaking table and to spread cultivation bacterial strain, return to zero, be cultured to the OD of seed liquor with nonvaccinated substratum under 150r/min, 30 DEG C of conditions
600reach 0.95 ~ 1.0, stand-by;
(3) ferment tank produces enzyme: the liquid amount of fermention medium by volume per-cent 60% loads in 10L fermentor tank, then the edible vegetable oil adding 10mL is as defoamer, is taken up in order of priority carries out that sky slake substratum is real to disappear by the using method of fermentor tank; By in the inoculum size access fermention medium of the by volume per-cent 3% of the seed liquor in step (2), keep tank pressure 0.1MPa in fermenting process, stir speed (S.S.) is 250r/min, controls air velocity 2.0m
3/ h, leavening temperature is 31 DEG C, and fermentation time is 60h;
(4) collection of crude enzyme liquid: closed cans discharging after fermentation, fermented liquid is centrifugal 10min under 3000r/min rotating speed, finally merges supernatant liquor, namely obtains the crude enzyme liquid containing endo-type chitoanase.
Carry out enzyme activity determination to the crude enzyme liquid of step (4) gained, result shows, in fermented liquid, the enzyme activity of endo-type chitoanase is 32.9U/mL.
embodiment 6
The preparation of colloid chitosan: chitosan is dissolved in the HC1 solution of 0.5mol/L with the concentration of 1g/L, room temperature for overnight, the NaOH solution of volumetric molar concentration such as to add, be neutralized to pH 10.0, flocks is filtered to obtain with 400 order yarn thin,tough silk, and by water repeated washing precipitation until pH is neutral, finally with the speed homogenized precipitation 12min of 10000r/mim, after beating, namely obtain colloid chitosan.
In actication of culture, slat chain conveyor based formulas used is: (NH
4)
2sO
40.65g, K
2hPO
40.35g, sodium-chlor 0.9g, magnesium sulfate 0.12g, colloid chitosan 1.08g, agar 2.0g, adds water to 100mL, pH 7.0.121 DEG C, sterilizing 20 minutes, stand-by.
Liquid seed culture medium formula is: peptone 1.0g, glucose 0.35g, sodium-chlor 0.8g, K
2hPO
41.0g, KH
2pO
40.05g, yeast powder 0.4g, magnesium sulfate 0.06g, adds water to 100mL, natural pH.200mL substratum loads 500mL triangular flask, seals with 8 layers of gauze, tin mulberry paper, puts into the Autoclave sterilizing 20 minutes of 121 DEG C, stand-by.
Fermentative medium formula is: colloid chitosan 1.0g, sodium-chlor 0.8g, K
2hPO
40.12g, KH
2pO
40.06g, magnesium sulfate 0.04g, yeast extract 1.5g, peptone 1.5g, adds water to 100mL, adjusts pH to 7.0 with 2mol/L hydrochloric acid.
Fermenting process comprises the following steps:
(1) actication of culture: adopt the method that line is separated, by the misty rose streptomycete preserved
streptomyces roseolusthe plate culture medium that it is sole carbon source that DH is inoculated in colloid chitosan, is inverted cultivation 2 days with activated strains in 30 DEG C of incubators;
(2) seed liquor is cultivated: the single colony inoculation after picking activation, in liquid seed culture medium, carries out shaking table and to spread cultivation bacterial strain, return to zero, be cultured to the OD of seed liquor with nonvaccinated substratum under 150r/min, 30 DEG C of conditions
600reach 0.9 ~ 0.95, stand-by;
(3) ferment tank produces enzyme: the liquid amount of fermention medium by volume per-cent 50% loads in 10L fermentor tank, then the edible vegetable oil adding 9mL is as defoamer, is taken up in order of priority carries out that sky slake substratum is real to disappear by the using method of fermentor tank; By in the inoculum size access fermention medium of the by volume per-cent 2.5% of the seed liquor in step (2), keep tank pressure 0.1MPa in fermenting process, stir speed (S.S.) is 200r/min, controls air velocity 1.5m
3/ h, leavening temperature is 30.8 DEG C, and fermentation time is 58h;
(4) collection of crude enzyme liquid: closed cans discharging after fermentation, fermented liquid is centrifugal 10min under 3000r/min rotating speed, finally merges supernatant liquor, namely obtains the crude enzyme liquid containing endo-type chitoanase.
Carry out enzyme activity determination to the crude enzyme liquid of step (4) gained, result shows, in fermented liquid, the enzyme activity of endo-type chitoanase is 31.3U/mL.
embodiment 7 endo-type chitoanase degrade chitosan prepares oligochitosan
In 1000mL beaker, add deionized water 500mL, add chitosan 25g under stirring, then add glacial acetic acid 7.5mL, continue to stir until be dissolved into colloid, pH value controls 5.0 ~ 5.5.Add embodiment 1 by the proportionlity of endo-type chitoanase vigor/chitosan=5U/g to ferment the crude enzyme liquid of gained, mixing, enzyme digestion reaction temperature controls at 45 DEG C, and the enzyme digestion reaction time is 5 hours, finally obtains enzymolysis solution with ultrafiltration membrance filter.Enzymolysis solution after ultrafiltration carries out spraying dry after concentrated, obtains oligochitosan product, and through high performance liquid chromatography qualification, the polymerization degree of oligochitosan product is 2 ~ 10.
High-efficient liquid phase chromatogram condition is:
Click maltose chromatographic column, 250 × 4.6 mm;
Light scattering detector, detector temperature 80 DEG C, detector air flow 1.5L/min;
The pH of damping fluid formic acid-ammonium formiate is 3.1, and concentration is 100mmol/L;
Chromatogram column temperature 30 DEG C, sample size 20 μ L, eluent flow rate 1.0mL/min;
Adopt gradient elution, specifically in table 1:
Table 1 gradient elution program (unit: volume percent)
。
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the prerequisite not departing from the inventive method; can also make some improvement and supplement, these improve and supplement and also should be considered as protection scope of the present invention.
Claims (10)
1. fermentable prepares a method for endo-type chitoanase, described microorganism be misty rose streptomycete (
streptomyces roseolus) DH, deposit number is CGMCC No.7742; It is characterized in that, described method comprises the following steps:
(1) actication of culture: by preserve misty rose streptomycete (
streptomyces roseolus) the DH plate culture medium that to be inoculated in colloid chitosan be sole carbon source, in 30 DEG C of incubators, be inverted cultivation 2 ~ 4 days with activated strains; Described slat chain conveyor based formulas is: (NH
4)
2sO
40.51 ~ 1.0g, K
2hPO
40.21 ~ 0.5g, sodium-chlor 0.51 ~ 1.0g, magnesium sulfate 0.01 ~ 0.2g, colloid chitosan 1.01 ~ 1.2g, agar 2.0g, adds water to 100mL, pH 7.0 ~ 7.2;
(2) seed liquor cultivate: picking activation after single colony inoculation in liquid seed culture medium, in 150r/min, 30 DEG C of conditions
Under carry out shaking table and to spread cultivation bacterial strain, be cultured to the OD of seed liquor
600reach 0.8 ~ 1.0, stand-by; Described seed culture based formulas is: peptone 0.51 ~ 1.0g, glucose 0.21 ~ 0.5g, sodium-chlor 0.8 ~ 1.0g, K
2hPO
40.02 ~ 1.0g, KH
2pO
40.01 ~ 0.05g, yeast powder 0.2 ~ 1.0g, magnesium sulfate 0.02 ~ 0.08g, adds water to 100mL, natural pH;
(3) ferment tank produces enzyme: the liquid amount of fermention medium by volume per-cent 40% ~ 70% loads in fermentor tank, by the seed liquor in step (2) by volume per-cent 2.1% ~ 3% inoculum size access fermention medium in, described fermentor tank is flat-blade turbine stirring-type fermentor tank, tank pressure 0.08 ~ 0.11MPa is kept in fermenting process, stir speed (S.S.) is 180 ~ 250r/min, controls air velocity 1.2 ~ 2.0m
3/ h, leavening temperature is 30 ~ 31 DEG C, and fermentation time is 50 ~ 60h; Described fermentative medium formula is: colloid chitosan 0.9 ~ 1.2g, sodium-chlor 0.8 ~ 1.0g, K
2hPO
40.05 ~ 0.15g,
KH
2pO
40.04 ~ 0.2g, magnesium sulfate 0.03 ~ 0.1g, yeast extract 0.2 ~ 2.0g, peptone 0.2 ~ 2.0g, adds water to 100mL, adjusts pH to 7.0 ~ 7.2 with 2mol/L hydrochloric acid;
(4) collection of crude enzyme liquid: closed cans discharging after fermentation, merges supernatant liquor after centrifugal fermented liquid, namely obtains the crude enzyme liquid of endo-type chitoanase.
2. prepare the method for endo-type chitoanase according to the fermentable described in claim 1, it is characterized in that, the product of described endo-type chitoanase hydrolyzing chitosan to be the polymerization degree be 2 ~ 10 oligochitosan.
3. the method for endo-type chitoanase is prepared according to the fermentable described in claim 1, it is characterized in that, the preparation method of described colloid chitosan is: chitosan is dissolved in the HC1 solution of 0.1 ~ 2.0mol/L with the concentration of 1g/L, room temperature for overnight, the NaOH solution of volumetric molar concentration such as to add, be neutralized to pH 9.0 ~ 10.0, flocks is filtered to obtain with 300 ~ 400 order yarn thin,tough silk, and precipitate 1 ~ 3 time with water repeated washing, until pH is neutral, finally with the speed homogenized precipitation 8 ~ 15min of 10000r/mim, namely colloid chitosan is obtained after beating.
4. prepare the method for endo-type chitoanase according to the fermentable described in claim 1, it is characterized in that, the slat chain conveyor based formulas in described step (1) is: (NH
4)
2sO
40.6g, K
2hPO
40.3g, sodium-chlor 0.52g, magnesium sulfate 0.1g, colloid chitosan 1.1g, agar 2.0g, adds water to 100mL, pH 7.0.
5. prepare the method for endo-type chitoanase according to the fermentable described in claim 1, it is characterized in that, the OD of seed liquor in described step (2)
600be 0.9 ~ 1.0.
6. prepare the method for endo-type chitoanase according to the fermentable described in claim 1, it is characterized in that, the seed culture based formulas in described step (2) is: peptone 0.6g, glucose 0.3g, sodium-chlor 0.8g, K
2hPO
40.07g, KH
2pO
40.03g, yeast powder 0.5g, magnesium sulfate 0.05g, adds water to 100mL, natural pH.
7. prepare the method for endo-type chitoanase according to the fermentable described in claim 1, it is characterized in that, the volume of fermentor tank is 10 liters in described step (3), and the liquid amount of fermention medium by volume per-cent 60% loads fermentor tank.
8. the method for endo-type chitoanase is prepared according to the fermentable described in claim 1, it is characterized in that, the inoculum size access fermention medium of seed liquor by volume per-cent 3% in described step (3), described stir speed (S.S.) is 200r/min, and air velocity is 1.4m
3/ h, leavening temperature is 30.5 DEG C, and tank pressure is 0.1MPa.
9. prepare the method for endo-type chitoanase according to the fermentable described in claim 1, it is characterized in that, the fermentative medium formula in described step (3) is: colloid chitosan 1.0g, sodium-chlor 0.8g, K
2hPO
40.07g, KH
2pO
40.04g, magnesium sulfate 0.05g, yeast extract 0.5g, peptone 0.5g, adds water to 100mL, adjusts pH to 7.2 with 2mol/L hydrochloric acid.
10. prepare the method for endo-type chitoanase according to the fermentable described in claim 1, it is characterized in that, the centrifugal condition in described step (4) is centrifugal 10min under 3000r/min rotating speed.
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| CN104926534A (en) * | 2015-06-29 | 2015-09-23 | 山东卫康生物医药科技有限公司 | High-purity chito-oligosaccharide controlled release fertilizer based on separation techniques and preparing method thereof |
| CN104945142A (en) * | 2015-06-29 | 2015-09-30 | 山东卫康生物医药科技有限公司 | Special chitosan oligosaccharide containing multi-trace-element foliar fertilizer for leaf vegetables and preparation method thereof |
| CN106434475B (en) * | 2016-11-01 | 2019-05-28 | 滕州市悟通香料有限责任公司 | One streptomycete category polysaccharide degradation bacteria and its cultural method and application |
| CN110156913A (en) * | 2019-05-21 | 2019-08-23 | 扬州日兴生物科技股份有限公司 | A method of discarded shrimp and crab shells chitin extraction is handled using bacillus cereus |
| CN111154788B (en) * | 2020-02-24 | 2022-03-08 | 中国科学院过程工程研究所 | Marine streptomyces nivalis chitosanase gene and application thereof |
| CN115786119B (en) * | 2023-02-13 | 2023-05-12 | 山东卫康生物医药科技有限公司 | Isolated culture control method and system for ginseng stem cells |
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